JP2024502033A - Specific detection of nucleic acid sequences using activation, cleavage and counting (ACC) technology - Google Patents

Abstract

The present disclosure provides a simple, single-step, room temperature activation, cleavage and counting (ACC) assay coupled to photonic resonator absorption microscopy (PRAM) in an amplification-free approach. The assays and related systems and methods disclosed herein enable the detection of viral and bacterial pathogens and diseases, such as cancer, in medical settings. [Selection diagram] Figure 1

Description

Since the SARS-CoV-2 (COVID-19) virus was transmitted from a reservoir animal to humans in December 2019, the virus has rapidly spread around the world, causing death, illness, disruption to daily life, and disruption to businesses and businesses. This results in economic loss for individuals. A key challenge for health systems in all countries is being able to diagnose this disease quickly and accurately, due to the limited number of test kits available and the limited number of accredited testing facilities. Coupled with the limited time required to obtain results and provide information to patients. Challenges associated with rapid diagnostic tests contribute to uncertainty about which individuals should be isolated, poor epidemiological information, and an inability to rapidly trace transmission within/between communities. The challenges underlying COVID-19 diagnosis are already well known from past encounters with emerging epidemics and pandemics, and challenges inherent in diagnosing mosquito-borne diseases (Zika, dengue, chikungunya, malaria), HIV, etc. He is also the representative of The ability to conduct widespread testing already provides countries that have done so, such as the Republic of Korea, with accurate information about who should be quarantined, thereby allowing for more timely control of disease transmission. It shows clear advantages that can be achieved. Continued surveillance is expected to continue even after the initial first wave of current COVID-19 infections, requiring travel, employment, and close person-to-person interactions. It is likely to be a more routine aspect of many situations.

However, the available techniques remain expensive (in terms of equipment capital equipment and reagents), technically difficult, and labor intensive. Therefore, there is an urgent need for a low-cost, portable platform that can provide rapid, accurate, and multiplexed diagnosis of infectious diseases in medical settings. Polymerase chain reaction (PCR) (Non-Patent Literature 1, Non-Patent Literature 2, Non-Patent Literature 3) and related approaches are characterized by the low amount of starting material (one genome copy per virus particle) and the instability of the RNA extraction process. , inhibitors in the test sample, and poor quality control of many reagents, resulting in high false-negative rates (Non-Patent Document 4, Non-Patent Document 5). Furthermore, enzymatic DNA/RNA amplification techniques are problematic when working from minimally processed samples in clinical settings, where false positives are a problem due to primer dimerization and perturbation of ideal buffer conditions. (Non-patent Document 6).

Additionally, disease detection has similar limitations. For example, cancer diagnosis requires expensive, complex, and time-consuming tests to accurately detect the presence of cancer. This places unnecessary physical and emotional strain on patients and contributes to increased healthcare costs.

C. Zhang and D. Xing, “Miniaturized PCR chips for nuclear acid amplification and analysis: latest advances and future trends”, Nucleic Acid. sRes. , Vol. 35, No. 13, pp. 4223-4237, 2007. J. Wang, Z. Chen, P. L. A. M. Corstjens, M. G. Mauk, and H. H. Bau, "A disposable microfluidic cassette for DNA amplification and detection", Lab Chip, Vol. 6, No. 1, pp. 46-53, 2006. S. H. Lee, S. -W. Kim, J. Y. Kang, and C. H. Ahn, “A polymer lab-on-a-chip for reverse transcription (RT)-PCR based point-of-care clinical diagnostics,” Lab Chip, Vol. 8, No. 12, 21. pp. 21-2127, 2008. “Types of Zika Virus Tests”, Center for Disease Control (CDC). [Online]. http://www. cdc. gov/zika/laboratories/types-of-tests. Available from html. R. N. Charrel, I. Leparc-Goffart, S. Pas, X. de Lamballerie, M. Koopmans, and C. Reusken, "Background review for diagnostic test development for Zika virus infection", Bull. World Health Organ. , Volume 94, Issue 8, Page 574, 2016. N. Bonne, P. Clark, P. Shearer, and S. Raidal, “Elimination of false-positive polymerase chain reaction results from hole punch carryover contamination”; J. Vet. Diagnostic Investig. , Vol. 20, No. 1, pp. 60-63, 2008.

Therefore, there is a need in the art to provide a reliable, rapid, inexpensive, low-cost, portable platform for the detection of infectious diseases and pathological conditions, such as cancer.

In one aspect, an exemplary embodiment provides a system for detecting nucleic acids in a sample. The system comprises a source substrate having streptavidin-linked nanoparticles attached to the surface of the source substrate by a nucleotide tether; an assay medium comprising a guide polynucleotide sequence and a Cas enzyme, wherein the guide polynucleotide sequence and the Cas enzyme target An assay medium capable of forming a CRISPR/Cas complex when exposed to a sample containing a nucleotide sequence; a biotinylated biosensor; and an imaging platform. The guide polynucleotide sequence is configured to bind to the target nucleotide sequence and a Cas enzyme, thereby forming a CRISPR/Cas complex, and the Cas enzyme is configured to cleave the nucleotide tether and release the streptavidin-linked nanoparticles. and an imaging platform configured to allow the streptavidin-conjugated nanoparticles to subsequently bind to the biotinylated biosensor and subsequently quantify the number of streptavidin-conjugated nanoparticles bound to the biotinylated biosensor. use.

In a further aspect, an exemplary embodiment provides a source substrate; a biotinylated biosensor; an assay medium comprising a guide polynucleotide sequence and a Cas enzyme; a population of streptavidin-conjugated nanoparticles; and a biosensor comprising a plurality of nucleotide tethers. The assay provides a biological assay in which streptavidin-conjugated nanoparticles are coupled to a biosensor using multiple nucleotide tethers, where the nucleotide tethers are comprised of nucleic acid sequences.

In another further aspect, an exemplary embodiment provides a method for detecting nucleic acids in a sample, the method comprising: binding streptavidin to nanoparticles to create streptavidin-containing nanoparticles. . Streptavidin-containing nanoparticles are attached to the surface of the source substrate using a nucleotide tether, thereby creating an assay surface. Biotinylated biosensors are produced by coating a biosensor with biotin. Activated Cas enzyme is produced by adding a test sample to an assay medium, the assay medium containing a guide polynucleotide sequence and a Cas enzyme, and the guide polynucleotide sequence and Cas enzyme containing a target nucleotide sequence. has the ability to form an activated CRISPR/Cas complex when exposed to. Streptavidin-containing nanoparticles that are cleaved upon incubation of the activated Cas enzyme and assay surface are subsequently captured and incubated with the biotinylated biosensor; the number of streptavidin-containing nanoparticles that bind to the biotinylated biosensor is determined by Quantify using an imaging platform.

In one aspect, an exemplary embodiment provides a system for detecting nucleic acids in a sample that includes a streptavidin-linked nanoparticle attached to a free-floating microparticle by a nucleotide tether. The system also includes an assay medium comprising a guide polynucleotide sequence and a Cas enzyme, the guide polynucleotide sequence and the Cas enzyme forming a CRISPR/Cas complex when exposed to a sample containing the target nucleotide sequence. can include assay media, biotinylated biosensors, and imaging platforms. In this system, a guide polynucleotide sequence binds to a target nucleotide sequence and a Cas enzyme, thereby forming a CRISPR/Cas complex, and the Cas enzyme cleaves the nucleotide tether, thereby releasing streptavidin-linked nanoparticles. is configured to do so. Subsequently, the streptavidin-conjugated nanoparticles are bound to the biotinylated biosensor, and the imaging platform is configured to quantify the number of streptavidin-conjugated nanoparticles bound to the biotinylated biosensor.

In a further aspect, a biological assay comprises streptavidin-linked nanoparticles, a free-floating microparticle, a biotinylated biosensor, an assay medium comprising a guide polynucleotide sequence and a Cas enzyme, a population of streptavidin-linked nanoparticles, and a plurality of nucleotides. Including tether. Streptavidin-linked nanoparticles are attached to free-floating microparticles using multiple nucleotide tethers, where the nucleotide tethers are comprised of nucleic acid sequences.

In another further aspect, a method for detecting a nucleic acid in a sample, the method comprising: conjugating streptavidin to a nanoparticle to create a streptavidin-containing nanoparticle; The method involves anchoring free-floating particles using tethers. Biotinylated biosensors are created by coating a biosensor with biotin. Activated Cas enzyme is produced by adding a test sample to an assay medium that includes a guide polynucleotide sequence and a Cas enzyme. The guide polynucleotide sequence and the Cas enzyme have the ability to form an activated CRISPR/Cas complex when exposed to a test sample containing the target nucleotide sequence, and upon incubation of the activated Cas enzyme cleaved streptavidin. The nanoparticles and free-floating microparticles are captured and the cleaved streptavidin-containing nanoparticles are incubated with the biotinylated biosensor. Streptavidin-containing nanoparticles that bind to the biotinylated biosensor are subsequently quantified using an imaging platform.

In one aspect, an exemplary embodiment provides a system for detecting nucleic acids in a sample. The system comprises a biosensor having nanoparticles attached to the surface of the biosensor by a nucleotide tether; an assay medium comprising a guide polynucleotide sequence and a Cas enzyme, wherein the guide polynucleotide sequence and the Cas enzyme target a target nucleotide sequence; an assay medium capable of forming a CRISPR/Cas complex when exposed to a containing sample; and an imaging platform. The guide polynucleotide sequence binds to the target nucleotide sequence and the Cas enzyme, thereby forming a CRISPR/Cas complex. The Cas enzyme is configured to cleave the nucleotide tether, thereby releasing the nanoparticle. The imaging platform is configured to quantify the number of nanoparticles tethered to the biosensor before and after sample addition.

In a further aspect, an exemplary embodiment provides a biological assay that includes a biosensor; an assay medium that includes a guide polynucleotide sequence and a Cas enzyme; a population of nanoparticles; and a plurality of nucleotide tethers. The nanoparticles are attached to the surface of the biosensor using multiple nucleotide tethers, which are comprised of nucleic acid sequences.

In yet another aspect, exemplary embodiments provide methods for detecting nucleic acids in a sample. Detection is achieved by tethering nanoparticles to the surface of the biosensor using nucleotide tethers, thereby creating an assay surface. Subsequently, assay medium is added to the assay surface. The assay medium includes a guide polynucleotide sequence and a Cas enzyme that are capable of forming a CRISPR/Cas complex when exposed to a sample containing the target nucleotide sequence. The method involves adding a biological sample potentially containing a target nucleotide sequence to an assay, thereby forming a CRISPR/Cas complex, and a biological sample tethered to a biosensor, before and after addition of the sample. Further comprising quantifying the number of nanoparticles using an imaging platform.

FIG. 1A is a schematic diagram of a portable PRAM detection instrument for illuminating a photonic crystal (PC) from below and depositing gold nanoparticles (AuNP) from above. FIG. 1B is a peak intensity value (PIV) image of the deposited AuNPs. Figure 1C shows the reduction in the resonant reflection intensity of PC with one AuNP. The concept of activation, cleavage and counting (ACC) assay is illustrated. The activated CRISPR/Cas RNP complex cleaves the DNA tether on the PC surface, leading to the detachment of the AuNP and causing a signal change. Figure 3A shows the promiscuous cleavage of the reporter gene caused by the RNP complex in the presence of 100 nM N gene. Figure 3B shows the promiscuous cleavage of the reporter gene caused by the RNP complex in the presence of 100 nM E gene. Figure 3C shows the increase in fluorescence emission from FAM after addition of 100 nM of each target gene. Figure 3D shows fluorescence emission intensity as a function of target N gene concentration. Figure 4A shows DNA tether cleavage and nanoparticle removal after addition of RNP complex targeting the N gene (100 nM) of the SARS-CoV-2 genome. Figure 4B shows DNA tether cleavage and nanoparticle removal after addition of RNP complex targeting the E gene (100 nM) of the SARS-CoV-2 genome. Changes in AuNP counts on PC before and after addition of control sample (inactivated Cas12a/gRNA complex) are shown for N and E genes, respectively. Figure 2 is a comparison chart depicting the relative changes of AuNPs in the presence of RNP complexes containing N gene, control (N gene), E gene and control (E gene), respectively. Figure 2 is a schematic diagram of AuNP capture by biotinylated PEG followed by activated cleavage and counting upon release of streptavidin-linked AuNPs and subsequent binding to a biotinylated biosensor. Figures 7A-7F are dose response curves from low concentration studies. Dose-response curves of bound AuNPs versus target concentration were shown at 0.1 aM (approximately 300 copies of target molecules in the test sample) (Figure 7A), 1aM (approximately 3,000 copies of target molecules in the test sample) (Figure 7B). ), 10aM (approximately 30,000 copies of target molecules in the test sample) (Figure 7C), 100aM (approximately 300,000 copies of target molecules in the test sample) (Figure 7D), and 1fM (approximately 30,000 copies of target molecules in the test sample) (Figure 7C), 3,000,000 copies of the target molecule) (FIG. 7E). The individual binding curves are plotted on a dose-response curve of bound AuNP versus target concentration (FIG. 7F). Continuation of Figure 7-1. Continuation of Figure 7-2. ACC dose response and target selectivity (1 of 2 data sets). Images of PC surfaces where AuNPs were captured after release by Cas12a following activation with EGFR gene fragments of (Fig. 8a) 1zM, (Fig. 8b) 10zM, (Fig. 8c) 100zM or (Fig. 8d) 1aM. Left and right panels show AuNPs captured using EGFR ^WT and EGFR ^L858R , respectively. (Figure 8e) Plot of AuNP counts from each image set (Figures 8a-8d). ACC dose response and target selectivity (2 of 2 data sets). Images of PC surfaces where AuNPs were captured after release by Cas12a following activation with EGFR gene fragments of (Figure 9a) 1zM, (Figure 9b) 10zM, (Figure 9c) 100zM or (Figure 9d) 1aM. Left and right panels show AuNPs captured using EGFR ^WT and EGFR ^L858R , respectively. (Figure 9e) Plot of AuNP counts from each image set (Figures 9a-9d).

It is to be understood that the particular aspects of the invention described herein are not limited to the particular embodiments presented, which may vary. It will also be understood that the terms used herein are for the purpose of describing particular embodiments only and are not intended to be limiting unless otherwise defined herein. Furthermore, as will be appreciated by those skilled in the art, certain embodiments disclosed herein can be combined with other embodiments disclosed herein without limitation.

Throughout this specification, unless the context indicates otherwise, the terms "comprise" and "include" and variations thereof (e.g., "comprises", "including") are used, unless the context clearly dictates otherwise. "comprising", "includes", and "including" include the listed component, feature, element, or step, or group of components, features, elements, or steps. , is understood to indicate not excluding any other component, feature, element, or step, or group of components, features, elements, or steps. Any of the terms "comprising," "consisting essentially of," and "consisting of" can be replaced by any of the other two terms while retaining their ordinary meaning.

As used herein, the singular forms "a," "an," and "the" refer to the plural unless the context clearly dictates otherwise. contains the referent of.

Unless the context clearly dictates otherwise, or the context and the understanding of one of ordinary skill in the art indicates otherwise, values herein that are expressed as ranges refer to ranges unless the context clearly dictates otherwise. Any particular value or subrange within the ranges described in the various embodiments of this disclosure, up to one-tenth of the lower limit of , can be envisioned.

As used herein and in the drawings, ranges and amounts can be expressed as "about" a particular value or range. "About" includes a precise amount. For example, "about 5%" also means "about 5%" and "5%." The term "about" can also refer to ±10% of a given value or range of values. Approximately 5% therefore also means, for example, 4.5% to 5.5%.

As used herein, "sample" refers to any type of sample containing nucleotide sequences, including biological samples. "Biological sample" includes, but is not limited to, an organ punch or tissue biopsy that is or may be afflicted with one or more of the diseases and/or disorders described herein. Refers to a sample of body tissue, or a sample of body fluid, including, but not limited to, blood, cerebrospinal fluid, plasma, or saliva from a warm-blooded animal, such as a mammal, preferably a human. Biological sample can also refer to tissue or blood samples obtained from non-human mammals and other animals.

In view of this disclosure, the methods and compositions described herein can be configured by one of ordinary skill in the art to meet desired needs.

1. Overview The present disclosure provides simple activation, cleavage and Clustered regularly spaced short palindromic repeats coupled to photonic resonator absorption microscopy (PRAM) in an approach that does not use enzymatic amplification of target nucleic acid sequences. An assay is provided by using sequence (CRISPR) based nucleic acid detection. The disclosed assays and detection devices can also be adapted to detect the presence of a wide range of infectious agents other than SARS-CoV-2, as well as pathological diseases, such as cancer. PRAM devices are described in U.S. Patent Application No. 16/170,111, while various aspects of photonic crystal (PC) biosensors are described in U.S. Pat. No. 521,769, No. 7,531,786, No. 7,737,392, No. 7,742,662, and No. 7,968,836, all of which are Incorporated herein by reference.

Microbial CRISPR and CRISPR-associated (CRISPR/Cas) adaptive immune systems contain programmable endonucleases that can be exploited for CRISPR-based diagnostics [7][8]. The systems, assays, and methods described herein utilize promiscuous single-stranded binding of these enzyme-guide RNA complexes (referred to as RNPs) to their specific targets (RNP activation). The ability to cleave nucleic acids is used to generate signal changes. However, current platforms require a pre-amplification step using sequence-specific primers and DNA polymerases in order to detect measurable changes with lateral flow test strips or fluorometers from the CRISPR process. In this disclosure, we utilize a PRAM biosensor imaging platform to perform rapid detection of specific target nucleic acid sequences to detect AuNPs attached to photonic crystal (PC) nanostructure surfaces with nucleic acid tethers [9] or Digital counting of nanoparticles containing streptavidin-linked AuNPs bound to a biotinylated biosensor is performed.

2. PRAM Working Principle A mobile version of the PRAM biosensing platform is shown in Figure 1A. Port 1 is coupled to a fiber-coupled 617nM LED light source (M617F2, Thorlabs) and a lens group (F810SMA-635, Thorlabs) is first utilized to collimate the output beam. A zero-order half-wave plate (WPH10M-633, Thorlabs) rotates the polarization of the collimated beam to excite the TM resonant mode of the PC cavity. A plano-convex lens (LA1509-A-ML, Thorlabs) then focuses the beam onto the back focal plane of an Olympus Plan Fluorite 20x/0.5 numerical aperture (NA) objective, from which the collimated beam hits the PC surface at normal incidence. A manual 3-axis stage (PT3, Thorlabs) is used to fix the PC sample in the focal plane of the objective lens. The reflected light from the PC resonator is collected by the same objective and redirected by a 50/50 non-polarizing beam splitter (CCM1-BS013, Thorlabs). A doublet (AC254-200-A-ML, Thorlabs) projects the image plane onto a charge-coupled device (CCD) camera (GS3-U3-51S5M-C, Point Gray) with a resolution of 177 nM/pixel. As shown in FIG. 1C, perfect interference occurs at a certain resonant wavelength and angle of incidence, and no light is transmitted, resulting in nearly 100% reflection efficiency. The magnitude of the resonant reflectance was dramatically reduced by the addition of absorbing AuNPs to the PC surface (Figure 1C), and as a result, each AuNP was can be observed (Figure 1B). Using a microscopy approach called photonic resonator absorption microscopy (PRAM), we measure the resonant peak intensity values (PIV) across the PC pixel by pixel, with the front side of the PC immersed in an aqueous medium, PIV images of the deposited AuNPs can be collected by illuminating the structure through the transparent substrate with standardized broadband light.

2. Assay Principle of Operation The Activation, Cleavage and Counting Assay (“Assay”) is a non-amplification biological assay with CRISPR-Cas based detection coupled to a PRAM biosensor imaging platform. In a first exemplary embodiment, the platform performs digital counting of streptavidin-conjugated gold nanoparticles (AuNPs) bound to a biotinylated biosensor. In a second exemplary embodiment, the platform provides digital counting of AuNPs released from a photonic crystal surface when a target nucleic acid sequence interacts with a guide polynucleotide sequence and Cas to form an activated complex. conduct.

The first embodiment of the assay is a biotinylated nanoparticle capture assay, in which a PRAM instrument is used to detect the number of streptavidin-conjugated nanoparticles that bind to a biotinylated biosensor. The biotinylated nanoparticle capture assay consists of a source substrate, a population of nanoparticles linked to streptavidin with an open pocket for biotin binding, an assay medium containing multiple nucleotide tethers, a guide polynucleotide sequence and a Cas enzyme, and biotin including chemical biosensors. As used herein, "open pocket" refers to one or more biotin binding sites on streptavidin that are available for biotin binding. More specifically, the source substrate contains a population of streptavidin-linked nanoparticles that are attached to the source substrate via multiple nucleotide tethers. The term "source substrate" refers to any biological material selected from materials including glass (silicon oxide), plastic (polyester, polystyrene, acrylic), metal (gold, silver), or dielectric (silicon nitride or titanium oxide). Refers to a chemically inert solid material. A source substrate is a surface that can hold nanoparticles in close proximity to its surface with one or more ssDNA tethers. In one embodiment, the source substrate is a PC biosensor.

Nanoparticles can be composed of a wide variety of materials. In one exemplary embodiment, the nanoparticles are gold nanoparticles (AuNPs). In other embodiments, the nanoparticle material is a quantum dot, a metal-based nanoparticle, a magnetic nanoparticle, or a nanoparticle composed of a dielectric material such as _SiO2 or _TiO2 . Magnetic plasmonic nanoparticle tags can also be used, whereby the biosensor binds to the nanoparticle by applying an attractive magnetic field between the released nanoparticle and the biotinylated biosensor. The required time can be shortened. Streptavidin-containing nanoparticles of the present disclosure are anchored to a source substrate using a DNA nucleotide tether comprised of non-specific nucleotide sequences. Consistent with this, the tether can be nearly any single-stranded DNA sequence. A portion of the tether may be dsDNA as shown in Figure 6, thereby providing rigidity and controlling the height displacement between the nanoparticle and the substrate. Nucleotide tethers may be uniform or heterogeneous in sequence and may be of unspecified length. In some embodiments, the nucleotide tether is about 5-200 nucleotides in length. In some embodiments, the tether is about 5-50, about 51-100, about 101-150, or about 151-200 nucleotides in length. In another further embodiment, the nucleotide tether has a length of about 5-25, about 26-50, about 51-75, about 76-100, about 101-125, about 126-150, about 151-175, or Approximately 176-200 nucleotides. Both ends of the tether can be prepared with chemical functionalities that facilitate the formation of covalent chemical bonds or biotin-streptavidin associations with the source substrate and the nanoparticles on the opposite ends of the tether.

In one exemplary embodiment, streptavidin is linked to the nanoparticles, preferably AuNPs, using pegylation or other methods known in the art for covalent or non-covalent attachment of streptavidin. attached. The second biotin binding site on streptavidin is utilized to bind to the nucleotide tether, thereby creating a nanoparticle-nucleotide tether-source substrate linkage as presented in FIG. 6. In one preferred embodiment, a tether, preferably ssDNA, anchors the streptavidin-linked AuNPs to the source substrate via an isocyanate. In alternative embodiments, ssDNA is tethered to streptavidin-linked nanoparticles, eg, AuNPs, via an alkyl halide, sulfonate, aldehyde, carboxylic acid, or epoxide.

The biotinylated nanoparticle capture assays disclosed herein detect the presence of one or more target RNA or DNA molecules whose sequence is a biomarker for a disease, the presence of a viral pathogen, or the presence of a bacterial pathogen. enable. Consistent with this, in one exemplary embodiment, a suspected nucleotide sequence having a target nucleotide sequence and therefore complementary to a guide polynucleotide sequence and capable of forming an activated CRISPR/Cas complex is provided. A sample and/or biological sample is incubated with a guide polynucleotide sequence and a Cas enzyme. The presence of target molecules in the sample results in activated Cas, and the concentration of activated Cas is directly proportional to the concentration of target molecules. A Cas-containing sample can contain both activated and non-activated Cas. Appropriate negative and positive controls can also be included in the reaction.

As used herein, a "Cas enzyme" can include any Cas enzyme that has the ability to form a Cas/CRISPR complex. Those skilled in the art will understand that Cas enzymes are classified into Class I and Class II. In one preferred embodiment, the Cas enzyme is a class II enzyme, more specifically Cas9, Cas12a, Cas12b, or Cas13a. However, CSN2, CAS4, CAS12C, CAS12D, CAS12E, CAS12E, CAS12G, CAS12H, CAS12I, CAS12I, CAS12I, C2C4, C2C8, C2C8, CAS13B, CAS13C, or CAS13D, or CAS13D. An alternative class II CAS enzyme that is not limited to these is also an assay It can be used as part of. In a preferred embodiment, Cas9 is used to detect messenger RNA, Cas12 is used to detect double-stranded DNA, and Cas13 is used to detect microRNA.

In the biotinylated nanoparticle capture assay, an activated Cas sample is incubated with a source substrate containing tethered streptavidin-linked AuNPs. Activated Cas cleaves the ssDNA tether, thereby releasing streptavidin-linked AuNPs into the assay medium. Assay medium containing streptavidin-linked AuNPs is incubated with the biotinylated biosensor, allowing streptavidin-biotin binding through the open pocket and subsequent quantification of streptavidin-biotin binding through the PRAM instrument. Quantitative changes in bound particles are indicative of the presence or absence of disease, viral pathogens, or bacterial pathogens in the sample or biological sample. The detection limit of the biotinylated nanoparticle capture assay is 1 zM for the target ctDNA sequence (Figures 7-9), which corresponds to approximately 3 copies of the target DNA sequence in the test sample. Three copies of the gene target suspended in 150 μL of solution is equal to 3.33×10 ⁻²⁰ M (33zM). Since clinical plasma samples are typically 5 mL in volume, 3 copies of the gene target in a 5 mL volume is equal to 1.00×10 ⁻²¹ (1 zM). Here, it is reported that the detection limit is 1 zM since the plasma DNA extraction kit elutes 100 μL of purified DNA from a maximum of 5 mL of human plasma. Therefore, the reported detection limit of 1 zM corresponds to the number of copies of the target gene present in 5 mL of plasma, so that the total DNA in the sample is separated in an elution volume of 100 μL and left for the subsequent cleavage step. A final dilution of 150 μL is assumed.

Preferably the biosensor is a photonic crystal. The biosensor can also be a whispering gallery mode biosensor that is a ring resonator, microtoroid, or microsphere. In further embodiments, the biosensor is a waveguide structure through which light travels laterally, an acoustic biosensor, a photoacoustic biosensor, or a surface plasmon resonance biosensor. In another further aspect, the AuNPs emitted from the source substrate are subsequently captured on a surface and subjected to other forms of microscopy, e.g., electron microscopy, dark field microscopy, or reflection microscopy, for counting the captured nanoparticles. Measured by interference microscopy. If the nanoparticles are photon emitters, such as quantum dots or phosphorescent nanoparticles, the microscopy system can be a fluorescence microscope or a total internal internal reflection fluorescence microscope. Figure 6 provides an overview of the AuNP capture assay.

Streptavidin-linked nanoparticles can also be tethered to micrometer-scale particles floating freely in a solution with activated Cas. As used herein, the term "particulate" refers to about 2 to 75 micrometers in diameter, about 2 to 70 micrometers in diameter, about 2 to 65 micrometers in diameter, about 2 to 55 micrometers in diameter, about 2 to 75 micrometers in diameter, 50 micrometers, approximately 2 to 45 micrometers in diameter, approximately 2 to 30 micrometers in diameter, approximately 2 to 25 micrometers in diameter, approximately 5 to 50 micrometers in diameter, approximately 5 to 35 micrometers in diameter, approximately 5 to 30 micrometers in diameter polymer beads ranging in size from about 5 to 25 micrometers in diameter, about 5 to 20 micrometers in diameter, about 5 to 15 micrometers in diameter, about 5 to 15 micrometers in diameter, or about 5 to 20 micrometers in diameter; Refers to microparticles that are magnetic beads or glass beads (silicon oxide). Free-floating micrometer-scale particles allow diffusion of microparticles and Cas in free solution, thereby allowing Cas to encounter more ssDNA tethers and cleavage of ssDNA in a shorter time. enable. In this embodiment, the use of free-floating microparticles requires an additional step after ssDNA cleavage, in which the microparticles are separated from the solution using centrifugation or a magnet, thereby separating the released microparticles in the supernatant. Isolate the streptavidin-conjugated nanoparticles. The supernatant containing streptavidin-conjugated nanoparticles was then incubated with a biotinylated biosensor to detect streptavidin-biotin binding through the open pocket and subsequent quantification of streptavidin-biotin binding via a PRAM instrument. enable. Quantitative changes in bound particles are indicative of the presence or absence of disease, viral, or bacterial pathogens in the sample or biological sample.

In a second embodiment, the assay disclosed herein is a method in which a CRISPR/Cas enzyme-guide RNA complex (referred to as RNP) is activated in a promiscuous manner after binding to its specific target (RNP activation). It operates on the principle of generating a signal change through its ability to cleave single-stranded nucleic acids. In this embodiment, AuNPs are attached to the surface of a photonic crystal (PC) via a DNA tether. Upon binding to specific SARS-CoV-2 RNA, the activated RNP complex non-specifically and repeatedly cleaves the DNA tether, releasing gold nanoparticles from the PC surface. A PRAM instrument then detects and counts the gold nanoparticles released on each surface, providing an immediate readout of the presence of SARS-CoV-2 RNA in the test sample, as shown in FIG. 2.

A second embodiment of a biological assay includes a biosensor; an assay medium comprising a guide polynucleotide sequence and a Cas enzyme, a population of nanoparticles; and a plurality of nucleotide tethers. The biosensor contains nanoparticles attached to a surface using multiple nucleotide tethers. Nanoparticles can be composed of a wide variety of materials, and in one embodiment, the nanoparticles are gold. In other embodiments, the nanoparticle material is a quantum dot, a metal-based nanoparticle, a magnetic nanoparticle, a nanoparticle composed of a dielectric material such as _SiO2 or _TiO2 , or a magnetoplasmonic nanoparticle. . The tether can be any RNA/DNA sequence.

Nanoparticles are tethered to the biosensor surface using nucleotide tethers composed of non-specific nucleotide sequences. In one embodiment, the source substrate is a PC biosensor. Nucleotide tethers may be uniform or heterogeneous in sequence and may be of unspecified length. In some embodiments, the nucleotide tether is about 5-200 nucleotides in length. In some embodiments, the tether is about 5-50, about 51-100, about 101-150, or about 151-200 nucleotides in length. In another further embodiment, the nucleotide tether has a length of about 5-25, about 26-50, about 51-75, about 76-100, about 101-125, about 126-150, about 151-175, or Approximately 176-200 nucleotides.

A biosensor can be a photonic crystal. The biosensor can also be a whispering gallery mode biosensor. Whispering gallery mode biosensors are ring resonators, microtoroids, or microspheres. In further embodiments, the biosensor is a waveguide structure through which light travels laterally, an acoustic biosensor, or a photoacoustic biosensor.

As mentioned above, a "Cas enzyme" as used herein can include any Cas enzyme that has the ability to form a Cas/CRISPR complex. In one preferred embodiment, the Cas enzyme is a class II enzyme, more specifically Cas9, Cas12a, Cas12b or Cas13a, but also Csn2, Cas4, Cas12c, Cas12d, Cas12e, Cas12f, Cas12g, Cas12h, Cas12i, Cas12k, Alternative class II Cas enzymes can also be used as part of the assay, including but not limited to C2c4, C2c8, C2c9, Cas13b, Cas13c, or Cas13d. In a preferred embodiment, Cas9 is used to detect messenger RNA, Cas12 is used to detect double-stranded DNA, and Cas13 is used to detect microRNA.

A second embodiment of the assay disclosed herein allows for the detection of the presence of a target RNA or DNA whose sequence is a biomarker for a disease, the presence of a viral pathogen, or the presence of a bacterial pathogen. Consistent with this, suspect samples and/or biological samples that carry the target nucleotide sequence and are therefore complementary to the guide polynucleotide sequence and have the ability to form an activated CRISPR/Cas complex are , added to the assay. Subsequently, the activated complex cleaves the nucleotide tether and the change in the number of bound nanoparticles is determined using a PRAM instrument. Quantitative changes in bound particles are indicative of the presence or absence of disease, viral, or bacterial pathogens in the sample or biological sample. More specifically, the quantitative difference is calculated as the difference between the nanoparticles tethered to the surface of the biosensor before and after addition of the sample. A decrease in the number of tethered nanoparticles is indicative of the presence of RNA or DNA molecules whose sequences are biomarkers for disease, viral pathogens, or bacterial pathogens. Furthermore, the nucleotide sequence of interest is also indicative of the presence of a disease. SARS-CoV-2 and cancer are exemplary viral pathogens and diseases that can be detected using the systems, assays, and/or methods disclosed herein. Following quantification of the tethered nanoparticles, the nanoparticles can be removed from the biosensor surface to allow reuse of the biosensor. Nanoparticles can be removed from the biosensor surface by exchanging the assay buffer or by agitating the assay buffer without exchanging the assay buffer. If nanoparticles are magnetic, they can also be removed from the biosensor surface by application of a magnetic field. The assays described herein can also be part of a system for detecting nucleic acids in a sample. The system of the present disclosure comprises a source substrate, a biosensor having nanoparticles attached to the surface of the biosensor by a nucleotide tether, or a biotinylated biosensor, a guide polynucleotide sequence configured to cleave the nucleotide tether, and a Cas enzyme. an assay medium comprising: a guide polynucleotide sequence and a Cas enzyme capable of forming a CRISPR/Cas complex when a sample containing a target nucleotide sequence is added to the assay; One or more of the PRAM imaging platforms are configured to quantify the number of nanoparticles tethered to the biosensor before and after addition.

The present disclosure also provides methods for using the assay in detecting nucleic acid sequences of interest in a sample, eg, nucleic acid sequences associated with an infectious agent, pathogen, or disease. A first exemplary embodiment is a method for detecting nucleic acids in a sample. Streptavidin is linked to the nanoparticles using pegylation or other techniques for conjugation that are commonly understood in the art. Streptavidin-containing nanoparticles are tethered to the surface of the source substrate using nucleotide tethers, thereby creating an assay surface and a biotin-coated biosensor. An assay medium is added to the assay surface, wherein the assay medium includes a guide polynucleotide sequence and a Cas enzyme, wherein the guide polynucleotide sequence and the Cas enzyme become activated when exposed to a sample containing the target nucleotide sequence. It has the ability to form CRISPR/Cas complexes. A biological sample potentially containing the target nucleotide sequence is added to the assay, thereby forming an activated CRISPR/Cas complex that releases streptavidin-containing nanoparticles. After release, a sample containing streptavidin-containing nanoparticles is added to the biotinylated biosensor, and an imaging platform is subsequently used to quantify the number of streptavidin-containing nanoparticles that bind to the biotinylated biosensor.

In a second exemplary embodiment of the assay, the nanoparticles of the present method are tethered to the surface of a biosensor using a nucleotide tether. Subsequently, assay media is added to the assay. The assay medium includes a guide polynucleotide sequence and a Cas enzyme that, when exposed to a sample containing the target nucleotide sequence or added to the assay, binds to the CRISPR/Cas complex. can be formed. Additionally, those skilled in the art will appreciate that the assay medium may also contain the necessary components to collect, store, or preserve the collected sample and/or biological sample. The sample and/or biological sample can be any sample suspected of containing nucleotide sequences. Those skilled in the art will appreciate that this includes, but is not limited to, tissue and/or body fluids from any mammal. In one preferred embodiment, the sample is of human origin. In some embodiments, the sample is from a non-mammalian host that may contain the target nucleotide sequence. Upon addition of a sample potentially containing the target nucleotide sequence to the assay, a CRISPR/Cas complex is formed and subsequently tethered to the biosensor using an imaging platform before and after addition of the sample. The number of nanoparticles collected is quantified. In one preferred embodiment, the imaging system is a PRAM imaging platform. The imaging platform can further include alternative non-imaging detection equipment. The imaging platform can also be a fluorescence microscope, a TIRF microscope, a dark field microscope, an electron microscope, an atomic force microscope, or a reflection interference microscope.

Additionally, a second embodiment provided herein enables the PRAM imaging system to rapidly detect the presence of a viral infection or disease when the concentration of the target molecule is high enough. It has the additional surprising technical effect of providing results in less than 20 minutes when used as part of the systems, assays, or methods disclosed in . As such, the systems, assays, and methods described herein can be used in medical settings.

The following examples are illustrative of specific embodiments of the invention and its various applications. They are included for illustrative purposes only and should not be construed as limiting the scope of the disclosure in any way.

Methods Silylation of PC Surfaces Surface functionalization was achieved using oxygen plasma to chemically activate the outer titanium oxide layer of the photonic crystal. Thereafter, the reactive hydroxyl groups generated by the plasma treatment were derivatized by liquid phase silylation using a silane mixture suspended in anhydrous tetrahydrofuran (THF) for 30 minutes at room temperature. The 50 mL solution consisted of 49 mL THF, 900 uL 3-(triethoxysilyl)propylisocyanate, 50 uL butyl(chloro)dimethylsilane, and 50 uL chloro(dimethyl)octylsilane. After silylation, the PC surface used to capture the cleaved AuNPs was immersed in an amine-containing solution at a concentration of 10 mg/mL in phosphate-buffered saline containing 0.5% N,N-diisopropylethylamine (DIPEA). It was subjected to secondary functionalization by reaction with PEG ₁₁ -biotin for 12 hours at room temperature.

AuNP surface treatment Amine/biotin-capped ssDNA tethers were incubated with streptavidin-conjugated gold nanoparticles (AuNPs) in 1 mM hydroquinone (suspended in nuclease-free water) at 30 °C for 30 min. Isolated after centrifugation at 200 rcf for 10 minutes. Subsequently, the ssDNA-bound AuNPs were resuspended in 1 mM hydroquinone and sonicated for 30 seconds.

Gold nanoparticle immobilization on PC surface ssDNA-conjugated AuNPs suspended in 150 uL of 1 mM hydroquinone buffer were immobilized on silylated PC surface by co-incubation for 30 minutes at room temperature. After immobilization, the PC surface was washed sequentially with four 50 mL aliquots of 1 mM hydroquinone.

Assembly of Cas12a-sgRNA complex Equal volumes of 100 nM enzyme (EnGen® Lba Cas12a) and 125 nM sgRNA were mixed together in 1× CutSmart® buffer (diluted in nuclease-free water). . This solution was incubated for 1 hour at 4°C to allow assembly of the Cas12a-sgRNA complex.

Targeted Activation of Cas12a-sgRNA Complexes The assembled Cas12a-sgRNA complexes are co-incubated with synthetic mutant dsDNA EGFR gene fragments for 2 minutes at 37°C in 150 uL of 1x CutSmart® buffer. It was activated by this. Each aliquot of activated complex was then added to a separate 1.5 mL Eppendorf centrifuge tube containing a 3 x 4 mm PC with immobilized AuNPs.

Surface cleavage and capture of AuNPs PCs immersed in target-activated Cas12a-sgRNA solution were incubated for 1 hour at 37°C and subsequently removed from the solution. The target activation solution containing the AuNPs released by cleavage was then transferred to another 1.5 mL Eppendorf tube containing the amine-PEG ₁₁ -biotin functionalized capture PC. Captured PCs were incubated for 1 h in a solution containing released AuNPs and washed before imaging.

Surface Imaging and Particle Counting After cleaning, the PC surface used for AuNP capture was irradiated with a 617 nM laser and imaged under a 50x microscope objective. Particle counts were measured after post-processing of the acquired images.

Example 1 Preliminary validation of CRISPR assay components in a fluorescence test demonstrates the ability of RNP complexes to cleave reporter sequences.
Preliminary validation of the CRISPR assay components was performed in the presence of a reporter sequence consisting of 6-carboxyfluorescein (6-FAM) at one end and black hole quencher (BHQ) at the other end. Activated RNP complexes for both compartments of the SARS-CoV-2 genome (denoted as N and E genes in Figure 3(A) and Figure 3(B), respectively) showed the ability to cleave reporter sequences. . As a result, in FIG. 3(C), a significant increase in the fluorescence emission intensity of the FAM reporter was observed. The same assay was repeated by varying the concentration of target N gene concentration. As shown in FIG. 3(D), when the target gene concentration was varied between 1 fM and 10 nM, no specific pattern in fluorescence intensity was observed.

Example 2 CRISPR assay reveals successful cleavage of DNA tethers in the presence of activated RNP complexes.
Following preliminary validation of the CRISPR components, the assay was performed on a PC using the principle shown in Figure 2. The detection assay consisted of the following components: (i) PC functionalized with AuNPs attached via a DNA tether, (ii) activated RNP complex: Cas 12a + guide RNA for defined target + target gene. , (iii) molecular biology grade water for washing the exfoliated AuNPs. Activated RNP complexes were added to polydimethylsiloxane (PDMS) wells, allowed to attach to PC, and incubated for 10 minutes. Thereafter, the wells were washed three times with molecular biology grade water. AuNP images before addition of activated RNP complexes and after washing with water were captured using portable PRAM. For both compartments of the SARS-CoV-2 genome, a decrease in AuNP counts was observed after addition of activated RNP complexes. This phenomenon led to the successful cleavage of the DNA tether linking the AuNPs on the PC surface in the presence of activated RNP complexes. Changes in the AuNP count results for the target N gene and the target E gene are shown in Figures 4(A) and 4(B), respectively.

The specificity of the assay was examined by comparing the cleavage activity of activated RNP complexes to that of non-activated complexes. The activated complex consisted of the target gene, whereas the non-activated complex (shown as a control in Figure 5 (A, B)) did not have the target gene in it. As observed in Figure 5(A), there was no significant change in AuNP counts before and after adding inactivated CRISPR complexes to PCs for both compartments of the SARS-CoV-2 genome. The chart shown in Figure 5(B) shows that approximately 95% of the AuNPs were removed in the presence of activated RNP complexes, whereas approximately 12% of the AuNPs were removed in the absence of the target gene in the control sample. Indicates that a change has been observed.

Example 3: Capture of nanoparticles released by Cas12a tether cleavage.
The capture-based assay involves targeted activation of RNP complexes, followed by on-boarding of individual 3 x 4 mm PCs in separate 1.5 mL tubes containing 150 μL of RNP complexes with known concentrations of the target gene. The cleavage of the ssDNA-tethered AuNPs found in Figure 1 was initiated by performing 1 hour at 37°C. After incubation, a 150 μL solution of RNP complexes, gene fragments and released AuNPs was transferred to a 200 μL volume 8×8 mm circular well containing a capture PC surface functionalized with biotinylated PEG. AuNPs suspended in 150 uL solution were incubated in wells containing capture PC for 1 hour, then removed from solution and imaged on a portable PRAM using a 50x objective. Negative control PCs incubated with RNP complexes containing no target gene fragment were imaged to measure AuNP counts associated with non-specific cleavage (background signal). Each captured PC incubated with samples containing RNP complexes and gene fragments, either EGFR ^WT (control) or EGFR ^L858R (mutant), was imaged to obtain AuNP counts. After subtracting the average background signal measured by imaging of negative control PCs, absolute AuNP counts for each PC were obtained for each of concentration (mol/L) and sequence identity (EGFR ^WT or EGFR ^L858R ). . Subsequently, their respective AuNP counts were used to generate dose-response curves for each gene fragment (control and mutant). No dose response was observed in the control samples, whereas in the case of the mutant gene fragments a linear dose response was achieved over the concentration range from 1 zM to 1 fM (FIGS. 7-9).

Conclusion The present disclosure demonstrates that detection of various compartments of the SARS-CoV-2 genome on a PRAM PC-based biosensing platform was successfully and rapidly performed. The flexible nature of CRISPR assay components makes the systems and assays described herein useful for the detection of infectious diseases and pathogens, as well as pathological conditions affecting human health, such as human cancer. , and strongly indicate the use of the method.

References
1. C. Zhang and D. Xing, “Miniaturized PCR chips for nucleic acid amplification and analysis: latest advances and future trends,” Nucleic Acids Res., vol. 35, no. 13, pp. 4223-4237, 2007.

2. J. Wang, Z. Chen, PLAM Corstjens, MG Mauk, and HH Bau, “A disposable microfluidic cassette for DNA amplification and detection,” Lab Chip, vol. 6, no. 1, pp. 46-53, 2006 .

3. SH Lee, S.-W. Kim, JY Kang, and CH Ahn, “A polymer lab-on-a-chip for reverse transcription (RT)-PCR based point-of-care clinical diagnostics,” Lab Chip, vol. 8, no. 12, pp. 2121-2127, 2008.

4. “Types of Zika Virus Tests.,” Center for Disease Control (CDC). [Online]. Available: http://www.cdc.gov/zika/laboratories/types-of-tests.html.

5. RN Charrel, I. Leparc-Goffart, S. Pas, X. de Lamballerie, M. Koopmans, and C. Reusken, “Background review for diagnostic test development for Zika virus infection,” Bull. World Health Organ., vol. 94, no. 8, p. 574, 2016.

6. N. Bonne, P. Clark, P. Shearer, and S. Raidal, “Elimination of false-positive polymerase chain reaction results resulting from hole punch carryover contamination,” J. Vet. Diagnostic Investig., vol. 20, no. 1, pp. 60-63, 2008.

7. GJ Knott et al., “Guide-bound structures of an RNA-targeting A-cleaving CRISPR-Cas13a enzyme,” Nat. Struct. Mol. Biol., vol. 24, no. 10, p. 825, 2017.

8. JS Chen et al., “CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity,” Science (80-. )., vol. 360, no. 6387, pp. 436-439, 2018.

9. TD Canady et al., “Digital-resolution detection of microRNA with single-base selectivity by photonic resonator absorption microscopy,” Proc. Natl. Acad. Sci., vol. 116, no. 39, pp. 19362-19367, 2019.

Claims (66)

A system for detecting nucleic acids in a sample, the system comprising:
a source substrate having streptavidin-linked nanoparticles attached to the surface of the source substrate by a nucleotide tether;
An assay medium comprising a guide polynucleotide sequence and a Cas enzyme, wherein the guide polynucleotide sequence and the Cas enzyme are capable of forming a CRISPR/Cas complex when exposed to a sample containing a target nucleotide sequence. medium;
Biotinylated biosensor;
including an imaging platform;
a guide polynucleotide sequence binds to a target nucleotide sequence and a Cas enzyme, thereby forming a CRISPR/Cas complex;
The Cas enzyme is configured to cleave the nucleotide tether, thereby releasing the streptavidin-linked nanoparticles;
Streptavidin-conjugated nanoparticles bind to biotinylated biosensors;
The imaging platform is configured to quantify the number of streptavidin-conjugated nanoparticles bound to the biotinylated biosensor system. 2. The system of claim 1, wherein the source substrate is a biologically inert solid material. 3. The system of claim 2, wherein the inert solid material is glass (silicon oxide). 3. The system of claim 2, wherein the inert solid material is a plastic composed of one or more of polyester, polystyrene, or acrylic. 3. The system of claim 2, wherein the inert solid material is gold or silver. 3. The system of claim 2, wherein the inert solid material is silicon nitride or titanium oxide. The system of claim 1, wherein Cas is Cas9, Cas12a, Cas12b, or Cas13. 2. The system of claim 1, wherein the target nucleotide sequence is indicative of SARS-CoV2. 2. The system of claim 1, wherein the target nucleotide sequence is indicative of cancer. 10. The system of claim 9, wherein the cancer is a solid tumor. 11. The system according to claim 10, wherein the cancer is a hematological cancer. The biosensor includes a photonic crystal, and the imaging platform includes a light source configured to excite resonance in the photonic crystal and a detector configured to detect light reflected from the photonic crystal. , the system of claim 1. 13. The system of claim 12, wherein the imaging platform is configured to quantify resonant peak intensity values measured pixel by pixel across the photonic crystal. 2. The system of claim 1, wherein the imaging platform includes non-imaging detection equipment. 2. The system of claim 1, wherein the biosensor is a whispering gallery mode biosensor. 16. The system of claim 15, wherein the whispering gallery biosensor is a ring resonator, microtoroid, or microsphere. 2. The system of claim 1, wherein the biosensor includes a waveguide structure in which light travels laterally. 2. The system of claim 1, wherein the biosensor is an acoustic biosensor. 2. The system of claim 1, wherein the biosensor is a photoacoustic biosensor. 2. The system of claim 1, wherein the biosensor is a surface plasmon resonance biosensor. Source board;
Biotinylated biosensor;
an assay medium comprising a guide polynucleotide sequence and a Cas enzyme;
A biological assay comprising: a population of streptavidin-linked nanoparticles; and a plurality of nucleotide tethers;
Streptavidin-linked nanoparticles are attached to biosensors using multiple nucleotide tethers, which are composed of nucleic acid sequences, for biological assays. 22. The biological method of claim 21, wherein a sample containing a detection nucleotide target sequence complementary to a guide polynucleotide sequence can be added to the assay to form an activated CRISPR/Cas complex. Target assay. 22. The biological assay of claim 21, wherein the Cas enzyme is Cas9 and the nucleotide target sequence for detection is messenger RNA (mRNA). 22. The biological assay of claim 21, wherein the Cas enzyme is Cas13 and the nucleotide target sequence for detection is a microRNA (miRNA). 22. The biological assay of claim 21, wherein the Cas enzyme is Cas12 and the nucleotide target sequence for detection is double-stranded DNA (dsDNA). The nanoparticles are gold nanoparticles, quantum dots, metal-based nanoparticles, magnetic nanoparticles, magnetoplasmonic nanoparticles, phosphorescent nanoparticles, or nanoparticles composed of dielectric materials such as SiO ₂ or TiO ₂ 22. The biological assay of claim 21. 22. The biological assay of claim 21, wherein the biosensor includes a photonic crystal and further includes an imaging platform configured to quantify resonance peak intensity values measured pixel by pixel across the photonic crystal. 22. The biological assay of claim 21, wherein the biosensor comprises a non-imaging detection device. 22. The biological assay of claim 21, wherein the biosensor is a whispering gallery mode biosensor. 30. The biological assay of claim 29, wherein the whispering gallery biosensor is a ring resonator, microtoroid, or microsphere. 22. The biological assay of claim 21, wherein the biosensor comprises a waveguide structure in which light travels laterally. 22. The biological assay of claim 21, wherein the biosensor is an acoustic biosensor. 22. The biological assay of claim 21, wherein the biosensor is a photoacoustic biosensor. 22. The biological assay of claim 21, wherein the biosensor is a surface plasmon resonance biosensor. 22. The biological assay of claim 21, wherein the nucleotide tether is comprised of a nucleotide sequence between 4 and 100 nucleotides in length. 36. The biological assay of claim 35, wherein the nucleotide tethers are uniform in nucleotide length. 36. The biological assay of claim 35, wherein the nucleotide tethers are non-uniform in nucleotide length. 22. The biological assay of claim 21, wherein the CAS enzyme is Cas9, Cas12a or Cas13a. 22. The biological assay of claim 21, which is stable at room temperature. A method for detecting nucleic acids in a sample, the method comprising:
conjugating streptavidin to the nanoparticles to create streptavidin-containing nanoparticles;
tethering streptavidin-containing nanoparticles to the surface of a source substrate using a nucleotide tether, thereby creating an assay surface;
coating the biosensor with biotin, thereby creating a biotinylated biosensor;
producing an activated Cas enzyme by adding a test sample to an assay medium, the step comprising:
The assay medium includes a guide polynucleotide sequence and a Cas enzyme;
the guide polynucleotide sequence and the Cas enzyme have the ability to form an activated CRISPR/Cas complex when exposed to a test sample containing the target nucleotide sequence;
capturing cleaved streptavidin-containing nanoparticles upon incubation of activated Cas enzyme and assay surface;
A method comprising: incubating cleaved streptavidin-containing nanoparticles with a biotinylated biosensor; and quantifying the number of streptavidin-containing nanoparticles that bind to the biotinylated biosensor using an imaging platform. 41. The method of claim 40, wherein the Cas enzyme is Cas9, Cas12 or Cas13. Claim: wherein the number of streptavidin-containing nanoparticles bound to the biotinylated biosensor is indicative of the presence of a target RNA or DNA molecule whose sequence is a biomarker for a disease, the presence of a viral pathogen, or the presence of a bacterial pathogen. 40. 41. The method of claim 40, wherein the target nucleotide sequence is indicative of SARS-CoV2. 41. The method of claim 40, wherein the target nucleotide sequence is indicative of cancer. 45. The method of claim 44, wherein the cancer is a solid tumor. 45. The method according to claim 44, wherein the cancer is a hematological cancer. The biosensor includes a photonic crystal, and the imaging platform includes a light source configured to excite resonance in the photonic crystal and a detector configured to detect light reflected from the photonic crystal. 41. The method of claim 40. 48. The method of claim 47, wherein the imaging platform is configured to quantify resonant peak intensity values measured pixel by pixel across the photonic crystal. 41. The method of claim 40, wherein the imaging platform includes non-imaging detection equipment. 41. The method of claim 40, wherein the imaging platform is a fluorescence microscope, a TIRF microscope, a dark field microscope, an electron microscope, an atomic force microscope, a reflection interference microscope. 41. The method of claim 40, wherein the biosensor is a whispering gallery mode biosensor. 52. The method of claim 51, wherein the whispering gallery biosensor is a ring resonator, microtoroid, or microsphere. 41. The method of claim 40, wherein the biosensor comprises a waveguide structure in which light travels laterally. 41. The method of claim 40, wherein the biosensor is an acoustic biosensor. 41. The method of claim 40, wherein the biosensor is a photoacoustic biosensor. Claims wherein the nanoparticles are gold nanoparticles, quantum dots, metal-based nanoparticles, magnetic nanoparticles, magnetic plasmonic nanoparticle tags, or nanoparticles composed of dielectric materials such as _SiO2 or _TiO2 . 40. A system for detecting nucleic acids in a sample, the system comprising:
Streptavidin-linked nanoparticles attached to free-floating microparticles by nucleotide tethers;
An assay medium comprising a guide polynucleotide sequence and a Cas enzyme, wherein the guide polynucleotide sequence and the Cas enzyme are capable of forming a CRISPR/Cas complex when exposed to a sample containing a target nucleotide sequence. medium;
Biotinylated biosensor;
including an imaging platform;
a guide polynucleotide sequence binds to a target nucleotide sequence and a Cas enzyme, thereby forming a CRISPR/Cas complex;
The Cas enzyme is configured to cleave the nucleotide tether, thereby releasing the streptavidin-linked nanoparticles;
Streptavidin-conjugated nanoparticles bind to biotinylated biosensors;
The imaging platform is configured to quantify the amount of streptavidin-conjugated nanoparticles bound to the biotinylated biosensor system. Streptavidin-linked nanoparticles;
Freely floating particulates;
Biotinylated biosensor;
an assay medium comprising a guide polynucleotide sequence and a Cas enzyme;
A biological assay comprising: a population of streptavidin-linked nanoparticles; and a plurality of nucleotide tethers;
Streptavidin-linked nanoparticles are attached to free-floating microparticles using multiple nucleotide tethers, where the nucleotide tethers are composed of nucleic acid sequences, for biological assays. A method for detecting nucleic acids in a sample, the method comprising:
conjugating streptavidin to the nanoparticles to create streptavidin-containing nanoparticles;
tethering streptavidin-containing nanoparticles to free-floating microparticles using nucleotide tethers;
coating the biosensor with biotin, thereby creating a biotinylated biosensor;
producing an activated Cas enzyme by adding a test sample to an assay medium, the step comprising:
The assay medium includes a guide polynucleotide sequence and a Cas enzyme;
the guide polynucleotide sequence and the Cas enzyme have the ability to form an activated CRISPR/Cas complex when exposed to a test sample containing the target nucleotide sequence;
capturing cleaved streptavidin-containing nanoparticles upon incubation of activated Cas enzyme and free-floating microparticles;
A method comprising: incubating cleaved streptavidin-containing nanoparticles with a biotinylated biosensor; and quantifying the number of streptavidin-containing nanoparticles that bind to the biotinylated biosensor using an imaging platform. A system for detecting nucleic acids in a sample, the system comprising:
a biosensor having nanoparticles attached to the surface of the biosensor by a nucleotide tether;
An assay medium comprising a guide polynucleotide sequence and a Cas enzyme, wherein the guide polynucleotide sequence and the Cas enzyme are capable of forming a CRISPR/Cas complex when exposed to a sample containing a target nucleotide sequence. a medium; and an imaging platform;
a guide polynucleotide sequence binds to a target nucleotide sequence and a Cas enzyme, thereby forming a CRISPR/Cas complex;
The Cas enzyme is configured to cleave the nucleotide tether, thereby releasing the nanoparticle;
The imaging platform is configured to quantify the number of nanoparticles tethered to the biosensor before and after sample addition to the system. Biosensor;
an assay medium comprising a guide polynucleotide sequence and a Cas enzyme;
A biological assay comprising: a population of nanoparticles; and a plurality of nucleotide tethers;
Nanoparticles are attached to the surface of biosensors using multiple nucleotide tethers, which are composed of nucleic acid sequences, for biological assays. A method for detecting nucleic acids in a sample, the method comprising:
tethering the nanoparticles to the surface of the biosensor using a nucleotide tether, thereby creating an assay surface;
adding an assay medium to the assay surface, the assay medium comprising a guide polynucleotide sequence and a Cas enzyme, the guide polynucleotide sequence and the Cas enzyme, when exposed to a sample containing the target nucleotide sequence, the CRISPR a step capable of forming a /Cas complex;
adding a biological sample potentially containing the target nucleotide sequence to the assay, thereby forming a CRISPR/Cas complex; and the number of nanoparticles tethered to the biosensor before and after addition of the sample. using an imaging platform. 63. The method of claim 62, wherein after quantification of the tethered nanoparticles, the nanoparticles are removed from the biosensor surface. 63. The method of claim 62, wherein the nanoparticles are removed from the biosensor surface by exchanging the assay buffer. 63. The method of claim 62, wherein the nanoparticles are removed from the biosensor surface by agitation of the assay buffer without assay buffer exchange. 63. The method of claim 62, wherein when the nanoparticles are magnetic nanoparticles, the nanoparticles are removed from the biosensor surface by application of a magnetic field.

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